ZMYND12 serves as an IDAd subunit that is essential for sperm motility in mice

Inner dynein arms (IDAs) are formed from a protein complex that is essential for appropriate flagellar bending and beating. IDA defects have previously been linked to the incidence of asthenozoospermia (AZS) and male infertility. The testes-enriched ZMYND12 protein is homologous with an IDA component identified in Chlamydomonas. ZMYND12 deficiency has previously been tied to infertility in males, yet the underlying mechanism remains uncertain. Here, a CRISPR/Cas9 approach was employed to generate Zmynd12 knockout (Zmynd12−/−) mice. These Zmynd12−/− mice exhibited significant male subfertility, reduced sperm motile velocity, and impaired capacitation. Through a combination of co-immunoprecipitation and mass spectrometry, ZMYND12 was found to interact with TTC29 and PRKACA. Decreases in the levels of PRKACA were evident in the sperm of these Zmynd12−/− mice, suggesting that this change may account for the observed drop in male fertility. Moreover, in a cohort of patients with AZS, one patient carrying a ZMYND12 variant was identified, expanding the known AZS-related variant spectrum. Together, these findings demonstrate that ZMYND12 is essential for flagellar beating, capacitation, and male fertility. Supplementary Information The online version contains supplementary material available at 10.1007/s00018-024-05344-7.


Introduction
The motility of sperm is vital for male fertility owing to the need for sperm to propel themselves along the length of the female reproductive tract following ejaculation so that they can fertilize the egg [1].Impaired sperm motility can directly result in male infertility [2].Asthenozoospermia (AZS) is a common type of primary male infertility wherein patients exhibit < 40% motile spermatozoa or < 32% progressive spermatozoa despite the absence of any abnormalities in sperm morphology or sperm counts [3].The genetic processes that govern sperm motility, however, remain incompletely understood.
As highly specialized cells, sperm consist of a head domain and a unique flagellum required for the oscillatory movement of these cells through the female reproductive tract such that they can fertilize mature oocytes [4].The flagellar structure is highly conserved and consists of the cytoskeletal axoneme and a range of specifically organized peri-axonemal elements [5].The axoneme is localized within the center of the flagella and is composed of nine peripheral doublet microtubules (DMTs) arranged around a central pair (CP) of microtubules in what has been termed a "9 + 2" arrangement [6].Microtubule dynamics are shaped and maintained through the links that are formed among dynein arms, radial spokes, and the nexin-dynein regulatory complex (N-DRC) [7].Variations in the many proteins present within sperm flagellum are thought to be closely associated with the pathogenesis of AZS in humans [8,9].The precise function of these proteins and how they contribute to mammalian infertility, however, has yet to be firmly established.
The swinging movement of sperm flagella is strongly dependent on dynein arm function, with axonemal dynein identification having yielded insight into the mechanistic basis for flagellar bending [10].Axonemal dyneins are complex molecular motors composed of heavy (DHC), intermediate (IC), light (LC), and light intermediate chain (LIC) polypeptides with various molecular weights and activities [11].Dynein motors are located within the axoneme, and are classified into the outer and inner dynein arms (ODAs and IDAs, respectively) [12].Any form of axonemal dynein arm defects in Chlamydomonas has been demonstrated to result in the severe impairment of ciliary motility [13,14].In both mice and humans, biallelic male sterility-related mutations in several genes related to dynein arm component biosynthesis including DNAH1 [15], DNAH2 [16], DNAH10 [17], and DNALI1 [1], have been identified.While affected patients exhibit reductions in sperm movement and multiple morphological abnormalities of the flagella (MMAF), the specific genetic basis for AZS is incompletely understood.ZMYND12 (Zinc Finger, MYND domain containing 12) is encoded on chromosomes 1 and 4 in humans and mice, respectively.The p38 homolog of ZMYND12 was first identified as an IDA component in Chlamydomonas [18].Seven IDA subspecies (a-g) have been identified to date, each of which consists of one or two of eight distinct DHCs [19].Prior studies identified p38 in Chlamydomonas as an IDAdspecific accessory subunit [20].Coutton et al. recently found that 3 of 167 patients with MMAF harbored variants in the ZMYND12 gene [21].In line with its possible functional role in this context, knockdown of the ZMYND12 ortholog TbTAX-1 in Trypanosoma brucei had a pronounced effect on sperm motility [21,22].These results suggest a potential role for ZMYND12 deficiency in human AZS.
Advances in gene editing-based models have enabled the in vivo investigation of the distinct phenotypic effects associated with knockout and knockdown models [23].Accordingly, a CRISPR/Cas9-based approach was herein used to generate Zmynd12-knockout mice as a means of exploring the phenotypic role played by ZMYND12 in vivo.

Deletion of the testis-enriched ZMYND12 results in male subfertility
Initially, murine ZMYND12 expression patterns across tissue types were analyzed via qPCR, revealing that it is expressed a high levels in the spleen and testis, with these levels being highest in the testis (Fig. 1A).Testis ZMYND12 mRNA levels initially began rising at 3 weeks postpartum and continued to rise with testis development into adulthood (Fig. 1B).When a STA-PUT (Sedimentation at Unit Gravity) approach was used for spermatocyte and spermatid isolation, the highest levels of Zmynd12 enrichment were detected in the round spermatids (RS) (Fig. 1C).Subsequent immunofluorescent staining confirmed that ZMYND12 was present in the flagella of elongated spermatids in the testes, in addition to being present within spermatozoa from both humans and mice (Fig. 1D; Supplementary Fig. S1).These results suggest that ZMYND12 may play an important functional role in the flagellum.
As a protein with a high degree of evolutionary conservation, human and mouse ZMYND12 exhibit high similarity (Supplementary Fig. S2).In an effort to understand its functional role in vivo, a CRISPR/Cas9 strategy was used to generate Zmynd12-knockout (Zmynd12 -/-) mice.For this approach, the zygotes of wild-type (Zmynd12 +/+ ) mice were microinjected with Cas9 and gRNAs targeting exons 2-7 of Zmynd12 (Fig. 1E).Subsequent PCR analyses confirmed the deletion of a 17,162 bp segment of the Zmynd12 gene in the resultant mice (Fig. 1F).Quantitative PCR additionally confirmed that the Zmynd12 was absent from the testes of Zmynd12 -/-mice, indicating that it had been successfully deleted (Fig. 1G).
The Zmynd12 -/-mice exhibited healthy growth with no evidence of any defects (Supplementary Fig. S3).When they were subjected to fertility testing, vaginal plus were detected, confirming the ability of adult Zmynd12 -/-males to mate with Zmynd12 +/-and wild-type females.Three males were used in each group, and each male was paired with two females.While the Zmynd12 -/-males were fertile, the average litter size was decreased, suggesting that loss of ZMYND12 can result in male subfertility (Fig. 1H).
Fig. 1 Sperm flagellin ZMYND12 deletion results in subfertility in male mice.(A) qPCR was used to detect the expression of Zmynd12 in murine samples prepared from different tissues, with 18 S as a normalization control.Data are means ± SEM, n = 3. (B) Zmynd12 mRNA levels in the testes of mice at different ages, with 18 S as a normalization control.W, weeks.Data are means ± SEM, n = 3. (C) qPCR was used to measure Zmynd12 expression in male germ cells isolated from the testes of mice, with 18 S as a normalization control.Data are means ± SEM, n = 3. (D) IF staining for ZMYND12 (red) and PNA (green) in the testes of WT mice, n = 3. (E) Schematic overview of the approach to generating Zmynd12 −/− mice using a CRISPR/Cas9 approach.(F) PCR was used to identify murine genotypes with the F1, R1, and R2 primers.Wildtype and knockout mice were respectively identified using the F1/R1 and F1/R2 primer pairs.(G) qPCR was used to measure Zmynd12 expression in the testes of Zmynd12 +/+ and Zmynd12 −/− mice, with 18 S as a normalization control.Data are means ± SEM, n = 3. (H) Average numbers of pups per litter for male Zmynd12 +/+ and Zmynd12 −/− mice.Data are means ± SEM, n = 3 pre-leptotene counts per tubule between these two groups of mice (Fig. 2D; Supplementary Fig. S4A).Furthermore, the flagella of elongated spermatids did not differ between the two genotypes (Supplementary Fig. S4B).Epididymal morphology was similarly normal in these Zmynd12 -/-mice (Fig. 2E).In addition, the Zmynd12 -/-mice were capable of producing sufficient morphologically normal spermatozoa (Fig. 2F-H).

ZMYND12 is essential for sperm motility
Given that it is an IDA component, ZMYND12 may serve as a regulator of flagellar motility.A computer-assisted Zmynd12 -/-mice exhibit normal spermatogenesis and spermatozoa morphology In an effort to explore drivers of subfertility in male Zmynd12 -/-mice, epididymal and testis samples from adult Zmynd12 -/-and control animals were analyzed.No differences in testicular size or appearance were observed when comparing these two groups of mice (Fig. 2A), nor was there any significant difference in the testicular/body weight ratio (Fig. 2B).Hematoxylin and eosin (H&E) staining revealed no evidence of apparent defects in Zmynd12 -/- testis sections (Fig. 2C), and there were also no differences in average spermatocyte, round spermatid, pachytene, or The flagella of ZMYND12-deficient sperm are structurally normal Flagellar structural integrity is vital for effective sperm motility.As such, transmission electron microscopy was used to evaluate the ultrastructural properties of sperm flagella from Zmynd12 +/+ and Zmynd12 -/-mice.The mid-piece of sperm from Zmynd12 -/-animals presented with the expected "9 + 2" axonemal microtubular arrangement, together with IDA, radial spoke, and outer dense fiber structures that were intact and consistent with those of Zmynd12 +/+ sperm (Fig. 4A-C).This suggests that Zmynd12 -/-spermatozoa do not present with any pronounced abnormalities, as was subsequently confirmed through immunofluorescent staining.
In Chlamydomonas flagella, the homolog of ZMYND12 is a structural component of the IDAd.However, analyses of several known DHCs and other IDA components revealed no abnormalities in any of these cases in samples from Zmynd12 -/-mice, even for the known IDAd component DNAH1 (Fig. 4D-I).These data suggest that the deletion of ZMYND12 does not alter the major structural characteristics sperm analyzer (CASA) was thus used to assess the motility of sperm from prepared knockout mice.While the spermatozoa of Zmynd12 +/+ mice were able to move in a linear manner, those of Zmynd12 -/-mice moved with a circular trajectory (Fig. 3A).In addition, the data revealed comparable total motility when comparing the samples from Zmynd12 +/+ and Zmynd12 -/-mice (Fig. 3B).However, these Zmynd12 -/-animals did exhibit significant reductions in sperm average path velocity (VAP), curvilinear velocity (VCL), and straight line velocity (VSL) as compared to controls (Fig. 3C-E).When these sperm were cultivated in a 37℃, 5% CO 2 incubator, the motility of Zmynd12 −/− sperm declined more dramatically relative to WT sperm (Fig. 3B-E).These results suggest a role for ZMYND12, with the loss of this protein potentially contributing to reduced sperm velocity.Immunoprecipitation was performed using anti-ZMYND12 or anti-IgG as a control (Fig. 5A; Supplementary Table S1).LC-MS/MS analyses of precipitates and western blotting identified PRKACA and TTC29 as candidate ZMYND12 binding partners when assessing only those targets interacting a minimum of three times (Fig. 5A-B).These interactions are partially consistent with results that have been reported in humans [21].
Immunofluorescent staining and western blotting for these candidate ZMYND12 binding partners were next performed on Zmynd12 +/+ and Zmynd12 -/-spermatozoa, of sperm flagella, indicating that ZMYND12 is an accessory IDA subunit the loss of which does not induce significant flagellar abnormalities in murine spermatozoa.

ZMYND12 influences the localization of PRKACA and regulates sperm capacitation
To further examine the possible mechanisms whereby ZMYND12 can regulate sperm motility, proteins extracted from the testes of adult WT mice were immunoprecipitated in an effort to identify ZMYND12-interacting proteins.90 min of in vitro capacitation.However, in contrast, the phosphorylation of Zmynd12 -/-spermatozoa showed significantly reduced compare to the controls (Fig. 5F).These results indicate that ZMYND12 may interact with TTC29 and PRKACA.As the ortholog of TTC29 in Chlamydomonas, p44, is an IDA component, the interaction between ZMYND12 and TTC29 in mice suggests functional conservation.Additionally, ZMYND12 loss reduced PRKACA levels in the flagellum.revealing a significant decrease in PRKACA and TTC29 levels in the absence of functional ZMYND12 (Fig. 5C-E), indicating that the absence of ZMYND12 may affect the assembly of these two proteins.PRKACA is a catalytic subunit of protein kinase A (PKA) known to serve as a regulator of sperm capacitation [24].Spermatozoa capacitation was next assessed.Western blotting using a specific anti-phosphotyrosine antibody indicated increased protein phosphorylation when wild-type spermatozoa underwent Fig. 5 ZMYND12 regulates sperm capacitation and engages in interactions with PRKACA.(A) IP was performed using wild-type mouse testis samples, after which silver staining and mass spectrometry were performed, revealing that PRKACA and TTC29 were possible interacting proteins.Exp, experimental group, in which interacting proteins were precipitated with an anti-ZMYND12 antibody; Ctl, control group, where an anti-IgG was used as a negative control for immunoprecipitation; Coverage, the coverage of the identified peptide relative to the protein; #Peptides, the types of peptides identified, n = 3. in mice thus fails to give rise to any apparent morphological defects impacting the brain ventricles and tracheal cilia.Additional research, however, will be necessary to characterize the functional performance of Zmynd12 −/− cilia.

Identification of biallelic ZMYND12 variants in a patient with AZS
Whole exome sequencing (WES) analyses led to the identification of a biallelic ZMYND12 variant in a patient with AZS (A001: II-1, 35 years old) (Fig. 7A).Through sanger sequencing, this biallelic ZMYND12 variant was confirmed to have originated from two asymptomatic heterozygous parents, suggesting that it is subject to autosomal recessive inheritance (Fig. 7B).This variant entailed a 1-bp insertion that was predicted to introduce a translational frameshift and a premature stop codon at position 38 of the 53th ZMYND12 amino acid coding sequence (Fig. 7C).Analyses of semen samples from this proband individual (A001) The brain and tracheal ciliary morphology of Zmynd12 -/-mice are normal The "9 + 2" microtubular arrangement is conserved in sperm flagella and in all motile cilia.Furthermore, defects in IDAd have been shown to cause ciliopathies, including primary ciliary dyskinesia (PCD) and MMAF in humans [15,25,26].Zmynd12 -/-mice were next evaluated for any potential PCD-related phenotypes.Initial analyses suggested that the brain samples of these knockout mice exhibited weights and external morphological characteristics consistent with those of Zmynd12 +/+ animals (Fig. 6A-B).Consistently, brain sections from these Zmynd12 -/-mice that had been stained with H&E appeared similar to those of WT mice (Fig. 6C).
Further comparisons of the tracheas of Zmynd12 -/-and WT mice revealed no differences in the length of tracheal cilia, nor were there any differences in the expression or localization of AC-TUBULIN in tracheal sections from these animals (Fig. 6D).The loss of ZMYND12 function

Discussion
In this study, ZMYND12 was identified as an IDA component in mice that is expressed at the highest levels in male germ cells and that is required for normal sperm motility and capacitation.Zmynd12 -/-male mice exhibited subfertility phenotypes, with reductions in sperm velocity despite the absence of any overt structural defects.The ability of ZMYND12 and TTC29 to interact supports their association with the IDAd subspecies in murine spermatozoa, and further analyses suggested that ZMYND12 may regulate sperm capacitation by influencing PRKACA assembly.

ZMYND12 serves as a specialized IDAd accessory subunit in mice sperm
Axonemal dynein consists of the ODA and IDA, with the ODA repeating every 24 nm and 7 IDA subspecies repeating in the 96 nm range [29].IDAs are important for bending motions, whereas ODAs provide acceleration [30].IDAs have a complex composition, with the major IDA species (a, b, c, d, e, f/I1, and g) exhibiting distinct compositional and location profiles within the axoneme [31].These IDAs are composed of multiple subunits that are broadly classified into the DHC, LC, IC, and accessory subunit categories [18].Distal DHCs play a vital role in the conversion of ATP-derived chemical energy into mechanical force, thereby driving ciliary motility [32].Proximal IC, LC, and LIC light subunits form the foundation of IDA complexes and are thought to regulate dynein activity.Of these, ICs are specific to IDA f/I1, while many other IDA components revealed a total motility of 7.25%, and a progressive motility of 1.5% (Table 1).In line with prior reports [21], this patient presented with a high proportion of morphologically abnormal sperm (Table 1).[27,28]; M, million Fig. 7 Identification of a ZMYND12 mutation in a male with AZS.(A) Pedigree analysis of the family affected by biallelic ZMYND12 variations.Males suffering from infertility are marked with filled black squares.(B) Sanger sequencing was used to verify ZMYND12 variants identified using whole-exome sequencing in a male with AZS (A001).Inserted bases are marked with a black dashed box (C) The locations of variations in the ZMYND12 gene association between MMAF incidence and ZMYND12 truncating and frameshift variants [21].In the present study, a novel variant in ZMYND12 associated with loss of function was identified in an MMAF patient with high sperm malformation rates.These results highlight the potential pathogenic effects of the loss of ZMYND12 as a driver of male infertility, extending the known spectrum of AZS causes and therefore providing potential benefits to the genetic counseling and healthcare management of individuals found to harbor this genetic variant.
With progressive advances in the understanding of the genetic basis for male infertility, a growing number of animal models have been developed to confirm and characterize the pathogenicity of certain variants in rodents and primates [39,40].The knockdown of the ZMYND12 ortholog TbTAX-1 in Trypanosoma brucei, has been reported to markedly alter flagellar motility in a manner akin to the changes evident in the sperm of males bearing homozygous ZMYND12 variants [21].While the male knockout mice in this study showed signficantly reduced sperm movement velocity, no corresponding reduction in the total fresh sperm motility was observed.Moreover the sperm from these Zmynd12 −/− mice did not exhibit any overt morphological abnormalities in contrast to the findings from the evaluated human patient.This may suggest that ZMYND12 plays distinct roles in the flagella of sperm from humans and mice.Interestingly, TTC29, as an interaction partner of ZMYND12, has also been found to function differently in humans and mice.Multiple case studies have demonstrated that loss of TTC29 leads to MMAF in humans, while TTC29-deficient mouse sperm showed only subtle morphological defects [41,42].As in Chlamydomonas, TTC29 and ZMYND12 may function as accessory units in IDAd in mouse sperm, while in human sperm, they play a structural role in the flagella [18].These differences reflect the functional differences in IDAd between human and mouse sperm flagella.In conclusion, the present data offer clear evidence that ZMYND12 is required for sperm motility and capacitation in mice.The multiple documented cases of human ZMYND12 variants also support a potential link between variations in this gene and the incidence of MMAF, which is a specific AZS subtype.At the molecular level, ZMYND12 was identified as a binding partner for PRKACA, and TTC29, serving as a key regulator of the functionality of murine flagella.

Animal care and ethics
Mice were housed under specific pathogen-free conditions in a controlled setting (50-70% humidity, 20-22 °C, 12 h are believed to perform non-IDA functions.For example, DNALI1 is an IDAd LIC subunit that is also a component of cytoplasmic dynamin and is involved in IMT [1,33].Here, ZMYND12 was selected as the component of interest, with its Chlamydomonas ortholog, p38, having previously been classified as an IDAd accessory subunit [18].While the precise functional role of p38 was not established, it was found to localize to the cilia and to play a role in axonemal IDAd docking [34].Here, TTC29 was identified as an interaction partner of ZMYND12.The finding that the ortholog of TTC29 in Chlamydomonas is an IDAd subunit [18] suggests that ZMYND12 may show IDAd localization in mouse sperm.

ZMYND12 serves as a regulator of PRKACA assembly that induces sperm capacitation
In contrast to their prior characterization as simple, rigid structures, sperm flagella are now understood to be highly complex organelles that contain an array of specialized enzymes.TSSK4, for example, is a Testis-Specific Serine/ Threonine Protein Kinase (TSSK) family protein found in the outer dense fibers where it controls the structural organization and motility of sperm through interactions with ODF2 [35].The fibrous sheath scaffold protein AKAP (A kinase anchoring protein) is capable of binding to protein kinase A (PKA) and particular subcellular substrates to protect the biophosphorylation reaction [36].The TSSK6 kinase and the DUSP21 phosphatase also exhibit periodic binding activity within the axoneme of murine spermatozoa [37].
Here, an interaction between ZMYND12 and PRKACA was detected such that ZMYND12 deletion resulted in a pronounced drop in PRKACA levels.This supports a role for ZMYND12 as a regulator of the assembly of PRKACA, potentially by serving as an anchoring site for the binding of this kinase, which is important for sperm capacitation.Consistently, a significant reduction in Zmynd12 −/− sperm capacitation was observed in this study, highlighting a novel function for IDAd.

ZMYND12 exhibits species-specific differences in functionality between mice and humans
To date, many different dynein-associated genes have been established as candidate factors related to male infertility characterized by impaired sperm motility.The loss of DNAH1 function was the first such variant that was conclusively identified as a cause of MMAF cases of AZS [15].More recently, studies have documented links between male fertility and a range of dynein proteins including DNAH2, with many variants in these genes having been linked to MMAF symptoms [38].A recent report documented an 1 3 uterus of pseudopregnant recipient female mice.The genotypes of offspring were then confirmed through PCR amplification with primers detailed in Supplementary Table S2.

qPCR
Trizol (Thermo Fisher Scientific, 15,596,026) was used to extract total RNA from each sample, of which 1 µg per sample was then reverse transcribed with the PrimeScript™ RT reagent Kit (Takara, RR036A) based on provided directions to generate cDNA.SYBR Green Master Mix (Vazyme, Q131) and a LightCycler480II system (Roche) were then used for all qPCR analyses performed with primers shown in supplementary Table S2, with 18 S rRNA serving as a reference control.

Western immunoblotting
After extracting proteins with RIPA buffer (Beyotime, P0013B) and quantifying their levels with a BCA Kit (Beyotime, P0012), equal protein amounts were separated via 10% SDS-PAGE and transferred to PVDF membranes.Blots were then blocked with 5% BSA (Sigma, v900933) in TBS for 2 h at room temperature, followed by overnight incubation with appropriately diluted primary antibodies (4 °C, overnight).Blots were blocked four times using TBST (15 min/wash), followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (2 h, room temperature.A chemiluminescence reagent was then used for protein band detection.

Study patients
The AZS patients tested in this study were recruited from the Ningbo Women and Children's Hospital.Participants with abnormalities in somatic chromosome karyotypes, genomic azoospermia factor deletions, serum sex hormone levels, and scrotal ultrasonography were excluded from the analysis.This investigation received ethical approval (approval no.EC2020-048) from the above institution and all subject provided written informed consent prior to the initiation of the study.All protocols were conducted in accordance with the Declaration of Helsinki and approved by the institutional ethics review board.

5′-G T A A G T C C A C A T A C C C A C A A A G G-3′ and gRNA2: 5′-G C T G C C A C G T C A G C C T A C A C A G G-3′
). Zyotes from C57BL/6 mice were simultaneously injected with these gRNAs and Cas9 mRNA, after which the embryos were transferred into the

Co-immunoprecipitation
RIPA buffer (1 mL; Beyotime, P0013C) was used to extract total testicular proteins, followed by centrifugation (40 min, 13,000 rpm).Supernatants were then collected, precleared for 1 h using 30 µL of protein A/G beads (Bimake, B23202) at 4 °C, and the lysates were then incubated overnight at 4 °C with appropriate antibodies.Protein complexes were then combined with 60 µL of Protein A/G magnetic beads, followed by a further 6 h incubation at 4 °C.Supernatants were then removed, and beads were washed with RIPA buffer 5 times, followed by the addition of SDS loading buffer.Samples were then boiled for 10 min at 95 °C and dentured proteins were separated by SDS-PAGE and detected with appropriate antibodies.As a negative control, rabbit IgG was also used for co-immunoprecipitation.

Histological and immunofluorescent staining
Sperm samples were fixed with 4% paraformaldehyde (PFA) for 10 min before spreading on slides.The slides were dried and then rinsed three times with PBS.For the preparation of paraffin-embedded sections, tissues were fixed using 4% paraformaldehyde or modified Davidson's fluid (MDF) for 48 h.Then, these samples were treated with a gradient of 70%, 80%, 90%, and 100% ethanol, a 1:1 mixture of ethanol and xylene, and pure xylene.After embedding these samples in paraffin, 5 μm sections were cut.Before staining, sections were deparaffinized and rehydrated.For H&E staining, these tissues were strained with hematoxylin and eosin staining solution.For IF staining, sections were treated with 10 mM citrate solution (pH 6) while heating for antigen retrieval.Both the sperm samples and sections were blocked using 1% BSA (Sigma, v900933), followed by overnight incubation at 4 °C with appropriate primary antibodies, washed, and treated for 2 h with secondary antibodies and Hoechst 33,342 at room temperature.Samples were then fixed using glycero, covered using glass coverslips, followed by imaging with an LSM980 confocal microscope (Carl Zeiss).

Transmission electron microscopy (TEM)
For TEM, samples were fixed with 1% osmium tetroxide and dehydrated with an ethanol gradient (50, 70, 90, and 100% ethanol) and 100% acetone.After infiltration with acetone and SPI-Chem resin and embedding with Epon 812, the samples were sectioned using an ultra-microtome and stained with uranyl acetate and lead citrate.A JEM-1400 transmission electron microscope (JEOL) was used for sample evaluation and imaging.
IgG, Alexa Fluor555 used for IF (Thermo Fisher Scientific, A-31,572), Donkey-anti-Rabbit IgG, Alexa Fluor488 used for IF (Thermo Fisher Scientific, A-21,206), and the anti-AKAP3 used for IF was a gift from Qi's lab [43].The working concentrations of the antibodies are shown in Supplementary Table S3.

Fertility testing
Fertility analyses were performed by mating sexually mature knockout male mice with two wild-type C57BL/6 female mice for a 6-month period during which the female mice were exchanged every other gestation cycle.Knockout male mice and controls were fed under identical conditions, and litter sizes were recorded during fertility testing.All fertility testing was conducted using 8 to 10-week-old mice.

Silver staining and LC-MS/MS
After separating proteins by 12% SDS-PAGE, they were stained with a Fast Silver Stain Kit (Beyotime, P0017S).Bands of interest were then excised manually, digested using sequencing-grade trypsin (Promega, WI, USA), and the peptides therein were extracted, dried, and analyzed via LC-MS/MS.
The IP precipitates were separated on SDS-PAGE and stained with AgNO 3 .The bands were removed from the gels following trypsin digestion.The EASY-nanoLC 1200 system (Thermo Fisher Scientific), equipped with an Orbitrap Q Exactive HFX mass spectrometer (Thermo Fisher Scientific) and a nanospray ion source, was used for LC-MS/ MS analysis.Mixtures of tryptic peptides were dissolved in 0.1% formic acid (FA) in LC-grade water and injected into an analytical column (75 μm× 25 cm, C18 column, 1.9 μm, Dr. Maisch).Solution A was 0.1% FA and solution B was 80% ACN and 0.1% FA.A 95-min linear gradient (3-5% B for 5 s, 5-15% B for 40 min, 15-28% B for 34 min and 50 s, 28-38% B for 12 min, 30-100% B for 5 s, and 100% B for 8 min) was applied using a high-resolution MS pre-scan, with a mass range of 350-1500.The normalized collision energy for elevated energy collision-driven dissociation (HCD) was adjusted to 28, and the resulting fragments were identified using a resolution of 15,000.All ions chosen for fragmentation were excluded for 30 s via dynamic exclusion.Data processing was done with Proteome Discoverer software (Thermo Fisher Scientific), and the mouse reference proteome was retrieved from the UniProt database (release 2021.04) using standard variables.

Table 1
Semen data of the patient